Production of dextran



United a.

PRODUCTION OF DEXTRAN Application October 30, 1956 Serial No. 619,362

2 Claims. (Cl. 195-31) (Granted under Title 35, US. Code (1952), sec. 266) No Drawing.

A non-exclusive, irrevocable, royalty-free license in the invention herein described, throughout the world for all purposes of the United Statese Government, with the power to grant sublicenses for such purposes, is hereby granted to the Government of the United States of America.

This application is a continuation-in-part of our copending application, Serial Number 348,134, filed April 10, 1953, now abandoned.

This invention relates to the preparation of dextran by direct biologic synthesis, and to a novel dextran product. It relates particularly to the biologic synthesis of a dextran possessing unusual molecular weight characteristics, by the reaction of Streptococcus sp. NRRL B-135-1.

It is known that carbohydrate gum, dextran, can be produced by the action of certain microorganisms on a sucrose containing medium. Heretofore the organisms used for commercial production of dextran have been various strains of L euconostoc mesenteroides. The individual strains vary as to the specific character of the dextran produced, each producing gums of different physical properties and different chemical structure. These differences, however, appear to vary in degree, with very few, if any, exceptions occurring among the various native dextrans produced by L. mesenteroides or by other known dextran producers.

The principal dextran product, and that customarily isolated directly from fermentation media, consistently has molecular weights greater than one million. We have recognized as accompanying the principal dextran product, though not generally isolated from the fermentation media, a second dextran component of average molecular weight consistently less than about 25,000. However, since dextran for use for pharmaceutical purposes, as blood volume expanders, and many other purposes, must be of an intermediate molecular weight, it has not been heretofore possible to produce them suitable for direct use.

Methods have been devised so that the proportion of low molecular weight dextran may be increased or decreased at the expense of high molecular weight dextran. Nevertheless, prior to our discovery, no significant amount of dextran of intermediate molecular weight has be known to occur as a direct fermentation product, and all attempts to vary the cultural conditions to produce such intermediate products directly from a simple sucrose containing medium have failed.

It has therefore been necessary to degrade the high molecular weight dextran by chemical, enzymic, or physical treatment in order to obtain dextran with weight aver age molecular weight of 25,000 to 200,000. High molecular weight dextran is conventionally hydrolyzed with acid, and the hydrolyzate carefully fractionated. Other'degradative methods that have been studied are pyrolysis,

es Patent Patented Sept. 29, 1959 We have discovered that a certain organism, biologically related to the known deXtran producers, is capable of producing directly a dextran having a molecular Weight within the range of 25,000 and 200,000. The cultural conditions required by the organism are essentially analogous to those of known deXtran producers, and the reasons why our organism produces an intermediate-sized product are not known.

This organism, Streptococcus sp. var. DS strain 50 NRRL B1351, belongs to the viridans group of streptococci. However, it difiers from the currently recognized species of this group, S. salivarius, S. mitis, S. bovis, S. rhermophilus, and S. equinus. The properties of the streptococcus of our present disclosure and of these viridansorganisms are given in Table 1. Physiological, serological and product comparisons of Streptococcus; sp. group H of Hare, J. Path. and Bact., 41: 495 (1935);; Streptococcus sp. var. DS I-Iehre and and Neill, I. EXptL. Med, 83: 147 (1946), and by Hehre, Bull. NY. Acad.. Med, 24: 543 (1948), and Streptococcus sanguis, White; of White and Niven, J. Bacteriol., 51: 717 (1946), are shown in Table la. The data shows that our organism is more closely related to Streptococcus sanguis than to Streptococcus sp. group H.

Native dextrans of high molecular weight, i.e., many' enzymolysis or degradation by means of ultrasonic treatment.

solutions of 40 percent concentration. The lower the molecular weight of the main fraction, the'higher the concentration of alcohol required to precipitate it. The methanol precipitation data on the dextran produced by Streptococcus sp. var. DS strain 50, Hehre, J. Biol. Chem., 222: 739 (1956), clearly indicate a unique product in that its molecular weight varies from a few thousand (-18 percent not precipitated at 65 percent methanol) up to $200,000 at the lowest concentration of methanol.

Another difference between Streptococcus sp. var. DS and S. sanguis should be noted. Although the former has been isolated from cases of bacterial endocarditis, it'is also known to occur in normal human throats. The

latter species has been found only in the blood stream of heart vegetation of endocarditis patients.

TABLE Ia Streptococcus sp. Streptococcus Streptococcus sp., sanguis Group H of Hare (42 strains) Greening on blood agar Oxidation of hemoglobin." l. Hemolytic colonies 1 Growth at 10 degrees (1... Growth at 45 degrees CL Growth 011 40% bile blood agar Growth in media plus 6.5% NaGl Arglnine hydrolyzed Sodium hippurate hydrolyzed Slime synthesis, 5% sucrose broth." Dextran pptn. at final methanol concentration of:

t On 5% horse blood agar, incubated 2 days under Anaerobic condiions.

2 Group H-at dilutions from BLOOD-40,000; Strept. 50 at 4,00010.000.

a Group H-at dilutions from 4,00010,000; Strept. 50 at 1,00010,000.

4 Physiological characteristics of 22 strains examined by Hehre and Niell; dextran pptn. data applies only to product from strain 50.

However, it would seem unwise to assign a species name .to the Streptococcus of our present disclosure until such time as the relationships between these organisms have been resolved.

age molecular weight of the last percent of the product was 18,600. The inherent viscosities of these two end TABLE I Stre t. 5 .var. Strept, D,S 50 hi RRL Strept. saliuarius Strept. 'n itis Strept. box/1's thermopllzlus Strept. equrm a 1. source blood of patient saliva, sputum in saliva, sputum in saliva, feces and milk and milk human and i with subacute "various pulmonary various'pulmonary intestinal conproducts. b0 Vll18 feces; cndocarditis. infections; apical infections; pus from tents of cattle; urine and nabsccsses of teeth; upper respiratory milk of cows; flammatory carious lesions of tract and sinuses; sometunes in exudates. teeth and intestinal blood and various human feces. tract. organs in subacute endocarditis. 2. blood agar greening no greening greening greemngsometimesno greening--- no greening. 3. dextra'n fbrntiationun lsometimes 4. levan lorma ion 5. starch hydroly usually 1 i r 7 i i E isa: hydro ys S m-id variable acid usually usually 7. 8, innlin acid acid var able i usually 9, fim-iihihnsp variahle usually According to the present invention Streptococcus sp. NRRL B-1351 also reported as Streptococcus var. DS, strain 50 is cultivated in an aqueous medium comprising up to percent or more sucrose and a source of assimilable nitrogen. Depending upon the temperature, concentration of sucrose, and other factors, the fermentation time may vary however from as short as two days up to about two weeks. The temperature of the culture medium should be maintained at about 35 to 39 C. for best results, and the pH at the start of the fermentation should be adjusted, if necessary, to within the range of 6 to ,8.

After completion of the fermentation the dextran may be recovered by dilferential alcohol precipitation. A convenient recovery method is (1) precipitating extraneous matter with 35 percent alcohol, (2) centrifuging to remove the precipitate, (3) precipitating the dextran in the supernatant with 65 percent alcohol (ethanol or methanol), .(4) redissolving the washed dextran in water and (;5) precipitating again with 65 percent alcohol. If .itis desired to fractionate the product, this may generally be done by the incremental addition of alcohol over the fractions were 0.521 and 0.084, respectively. An 80 gram portion of this product was further fractionated with methanol at 25 C. in a 5 percent aqueous solution adjusted to pH 7. Three fractions, I, II, and III, were separated between the methanol concentration limits of 0 to 43 percent, 43 percent to 50 percent, and 50 percent to 65 percent with yields of 17 percent, 49 percent, and 21.5 percent of the original portion. The molecular weights of fractions 1, II and III were 199,000, 54,300 and 11,200, respectively.

Fraction II was further fractionated with methanol to determine the distribution of molecular weights of its components. The fraction (12.8) percent of highest molecular weight as separated by methanol precipitation had a weight average molecular weight of'96,700. The fraction (15.0 percent) of lowest molecular weight had a weight average molecular weight of 21,500.

Interpretation of the formic acid produced and the periodate consumed in a sodium periodate oxidation of fractions 1, II, and III indicate the following distribik tion of glucosidic linkages.

range of 42 to 50 percent. TABLE H The following specific examples illustrate the invention. V I V Linkage Example 1 Fraction 1-6 1-4 1-3 A culture medium (3 l.) was made up having the following composition. 9 9 iii" 53% i2 12% Sucrose '20 gum/100 ml. Tryptose 1.0 gm./-100 ml. Ex I 2 Yeast extract 0.5 gm./ 100 ml. 55 e KaHPO, {L75 gm/LOOQL The following example illustrates the behavior of the The medium was adjusted to pH 7.6, and following sterilization, was inoculated with Streptococcus ,sp. NRRL .B-1351. The temperature was maintained at about 37 C. for a period of 72. hours, at whichtime the medium was centrifuged, neutralized-with sodiurn hydroxide and clarified. The supernatant liquor was made up to '65 percent ethanol, and the precipitate collected centrifugally. The precipitated material was dissolved in water. An insoluble material was obtainedbycentrifugal separation in the amount of 4.75 grams. The dissolved dextran was precipitated again at percent ethanol to yield 117 grams dry product. The weight average molecular weight of this product was 58,200 as determined by light scattering methods. Its inherentviscosity was 0.243. i i

The weight average molecular weight of the first 15.3 p ent h e ed ec a ata on thesras aate dd ti n .Of ethan 18 009 a d th mi ht eve organism in a culture medium containing a vditferent source of assimilable nitrogen. The medium employed was as follows:

Sucrose 200 gm./1000 1 1 1. Corn steep liquid solids 10 gm./l000 KH PQ, 10 gm./ 10001111.

Comparative fermentations were carried out to determine the efiect of sucrose concentration upon dextran production. The medium used in run A was made up as follows:

Sucrose 100 gm./1000 ml. Yeast protein digest gm./1000 ml. K HPO 20 gm./ 1000 ml. initial pH 7.6

The medium used in run B was the same as in run A except that the sucrose level was increased to 200 gm./ 1000 ml. The fermentation conditions were the same as in the still group of Example 2. Dextran determinations were made at the time intervals indicated in Table IV.

TABLE IV- Dextran Yield, percent of theory Time, Hrs.

Run A Bun B Analysis of the dextran fractionated from the 72 hour product of run A by a single precipitation between the 6 limits of and 52 percent methanol showed the following properties.

Light scattering mol. wt.

The reducing power molecular weight being higher than the light scattering molecular weight indicates the absence of reducing and groups customarily found for dextran prepared by hydrolysis and fractionation.

Dextran was fractionated from the 120-hour product of run B by high speed centrifugation to separate a small amount of very high molecular weight dextran followed by precipitation at percent methanol. The light scattering molecular weight was determined to be 60,600, and the yield based on the total dextran present was 40.7 percent.

We claim:

1. The method of producing dextran of intermediate molecular weight which comprises cultivating Streptococcus sp. NRRL B-1351 in an aqueous medium comprising sucrose as a chief source of assimilable carbon, and a source of assimilable nitrogen, continuing the fermentation until a substantial proportion of the sucrose has been converted to dextran by the organism, and recovering dextran from the culture medium.

2. The method of producing dextran of intermediate molecular weight which comprises culturing Streptococcus sp. NRRL B-l351 in an aqueous medium comprising approximately 10 to 20 percent sucrose and a source of assimilable nitrogen at an initial pH within the range of 6 to 8 for from two days to about two weeks and recovering dextran from the culture medium.

American Chem. Soc., Abstract of Papers, vol. 122, page 18A (1952). 

1. THE METHOD OF PRODUCING DEXTRAN OF INTEMEDIATE MOLECULAR WEIGHT WHICH COMPRISES CULTIVATING STREPTOCOCCUS SP. NRRL B-1351 IN AN AQUEOUS MEDIUM COMPRISING SUCROSE AS A CHIEF SOURCE OF ASSIMILABLE CARBON, AND A SOURCE OF ASSIMILABLE NITROGEN, CONTINUING THE FERMENTATION UNTIL A SUBSTANTIAL PROPORTION OF THE SUCROSE HAS BEEN CONVERTED TO DEXTRAN BY THE ORGANISM, AND RECOVERING DEXTRAN FROM THE CULTURE MEDIUM. 